The incidence of melanoma is increasing rapidly in the VA population in the United States, and sun exposure during US military service has been linked to increased melanoma incidence. The standard of care for advanced melanomas includes molecular-targeted therapies of BRAF and MEK inhibitors, as well as immunotherapies with checkpoint inhibitors such as anti-PD1. However, between 25% to 70% of advanced melanoma patients either do not respond or relapse from the current treatments. Therefore, discovering effective new therapies for malignant melanoma is still a pressing issue in the VA patient care mission. BH3 mimetics are a potent new class of cancer treatments that inhibit the BCL-2 dependent anti-apoptotic defenses inherent in cancer cells. Standard care plus an inhibitor against BCL-2 (Venetoclax) gave a response rates as high as ~80% in hematological malignancies. However, treatment with a single BH3 mimetic is insufficient to kill solid tumors, likely due to the presence of uninhibited BCL-2 family members. MCL-1 inhibitors, currently in clinical trials, can potentially boost treatment effectiveness when paired with clinically established BH3 mimetics. Further, most studies with BH3 mimetics focus on their ability to kill tumor cells directly, while their potential to enhance immunotherapies remains to be explored. The objective of this project is to test the combination treatment of MCL-1 inhibitors with other BH3 mimetics or with immunotherapy (anti-PD-1) in the treatment of melanoma, especially for melanoma tumor cells unresponsive to current treatments. Our preliminary data have indicated that MCL-1 inhibitors, when combined with ABT-263, can synergistically kill both the bulk of melanoma tumor cells and the melanoma initiating cells (MICs). The melanomas included those relapsed from current therapies. Further, in an immune competent model for melanoma (B16 in C57BL6 mice), a MCL-1 inhibitor increased the efficacy of anti-PD1 therapy (Preliminary Studies). We hypothesize: 1) MCL-1 inhibitors, as part of combination therapies, can overcome the resistance of melanoma patients to current treatments. 2) This elimination of tumor cells can be achieved either through direct killing or through modulating the antitumor immune response of tumor microenvironment. We also have access to a large collection of melanoma short-term cultures and patient-derived xenograft (PDX) models from the University of Colorado Melanoma Tissue Bank. Most of these materials were derived from melanomas of patients who had relapsed from the current treatments, with whole exosome and RNA-seq data available; they will provide the unique opportunity to test our hypotheses. Aim 1 will determine the mechanism(s) at play in the combination of MCL-1 inhibitors and ABT-263. We will further test the hypothesis that the combination of multiple BH3 mimetics, targeting MCL-1 plus other pro-survival BCL-2 family members, will overcome the resistance of melanoma cells to current therapies, in vitro and in vivo. Aim 2 will determine the efficacy and mechanisms of MCL-1 inhibitors to potentiate immunotherapies. We will test the hypothesis that MCL-1 inhibitors potentiate immunotherapies through blocking suppressive immune cell populations and increasing antitumor immunity in immune-competent mouse models. In summary, this proposal extends our promising preliminary studies using MCL-1 inhibitors as partners in drug combinations designed to overcome melanoma resistance to standard therapies. The expected outcome of exploring these new clinical-trial-ready BH3 mimetics will likely improve patient outcomes, especially for patient groups in dire need for alternative strategies. Results will likely lead to a positive impact on melanoma treatments, identifying new therapies for treating melanoma patients who have exhausted their other options.